The whole plant nuclear targeting assay, an experimental system that utilizes a vector derived from the cytoplasmic virus Potato virus X (PVX) to study and ultimately identify the nuclear targeting signals of Potato spindle tuber viroid (PSTVd), was developed by Zhao et al. (MPMI 82: 1911-1497, 2001). In that system, the coding region of the green fluorescence protein (GFP) was interrupted by insertion of an intron derived from the intervening sequence 2 of the potato ST-LS1 gene. In our study, four fragments representing different portions of the PSTVd upper RNA strand (1-180 bp) were embedded within the intron and the reporter RNAs were delivered into Nicotiana benthamiana plants via transient expression from a PVX-based vector. Plant responses among individuals to the same construct differed, including the time of appearance and the pattern of GFP lesions and the splice sites and efficiency of RNA splicing. This study demonstrated significant effects of the plant genome upon such conservative molecular mechanisms as RNA trafficking, splicing, and protein expression. In addition to the correctly spliced RNA, alternative splicing or RNA recombination occurred in individual plants, as revealed by RT-PCR and sequence analysis of products derived from the same construct containing certain small PSTVd fragments.

A variety of Phytophthora species have been associated with soils in oak regions of Europe and Turkey. A survey of oak forests in nine states (Indiana, Illinois, Maryland, Michigan, Minnesota, Pennsylvania, Ohio, West Virginia and Wisconsin) has been conducted to investigate the Phytophthoras present in forest soils. Samples taken from around the base of healthy and declining oak trees were baited with 3-7 day-old-oak leaflets and then plated on PARPNH- medium. P. cinnamomi was the most frequently isolated species representing 73.4% of isolates recovered. Other species included P. citricola, P. europaea, P. cambivora and a yet unidentified Phytophthora species. In total, 133 sites have been surveyed with 54.1% of sites yielding at least one Phytophthora species, and twenty-one percent of 820 soil samples proved positive for Phytophthora. These results suggest that numerous Phytophthora species are common to oak forest soils.

Sudden oak death (SOD) has emerged as a new concern for Maryland residents. For SOD, HGIC partnered with the Maryland Department of Agriculture to provide the public outreach. A critical issue such as SOD requires a rapid response to educate the public. Web-based materials as well as traditional methods can provide critical information quickly to individuals on a state and regional level.The objective was to quickly provide detailed information on biology, importance, photographs and IPM strategies on dealing with this issue. A sudden oak death web page was constructed on HGIC’s web site. The page provides up-to-date information on the SOD issue, links, sample submission forms and photo gallery. It received 5,044 visitors in 2004. The URL is: http://www.hgic.umd.edu/sod/sod_menu.html. Trained HGIC staff screened phone calls and email requests for SOD samples and provided MDA with a daily list of residents with potentially infected plant material, forwarding a total of 353 names.

The expense of regularly timed fungicide applications combined with the risk of resistance development and an increase in consumer preference for fruit produced with minimal pesticide inputs are prompting pumpkin growers in the Eastern United States to seek alternative management options. Many have employed no-till winter cover cropping systems. In 2002, Everts found that Plectosporium blight and black rot were significantly reduced for pumpkins grown on a no-till cover crop of hairy vetch or hairy vetch and rye, in contrast to conventional tillage and bare ground. However in one season, Fusarium fruit rot incidence was greater in the cover crop treatments. In order to determine the mechanism(s) by which disease is influenced by cover crops, experiments are being conducted on pumpkins produced in no-till rye, hairy vetch, rye plus hairy vetch, spring oat and bare ground systems. The studies examine the effects of chemical, physical, nutrient and water content changes, microclimate alteration, and microbial population shifts on disease development. Differences, including those in soil-surface residue, splash dispersal, fatty acid analysis, and soil nutrient content are being examined for their relationship to pumpkin fruit rots. A greater understanding of these relationships should lead to improved management strategies within a sustainable cropping system.

Many vegetable and field crop producers in Maryland grow pumpkins to diversify their operations and provide an alternative source of revenue. Studies were conducted to examine the use of host resistance and reduced risk fungicides, or cover crops on the management of foliar disease. A reduced-risk fungicide program based on the biofungicide Serenade and azoxystrobin or pyraclostrobin plus boscalid, which are classified as reduced risk fungicides, performed well compared to a conventional fungicide program in 2003, when disease pressure was low to moderate, but not in 2004, when downy mildew onset was early and severity was high. Some experimental lines and the commercial cultivar Harvest Time developed low or moderate level of downy mildew in comparison to other cultivars; Harvest Time may have a low to moderate level of resistance. A second study was conducted on pumpkins grown on one of ten cover crops or on bareground. The cover crops were killed in the spring and no-tilled. Downy mildew severity differed among pumpkins grown on the cover crop systems, and did not appear to be related to plant vigor. Host resistance in combination with reduced risk fungicide programs and cover crops comprise an integrated disease management system for pumpkin growers.

Production losses and loss of quality due to pests and diseases is a serious problem in cocoa (Theobroma cacao) production. In countries where cocoa is an important cash crop for small farmers, Integrated Pest Management (IPM) strategies that are reliable and economically viable are seen as the best approach. Until better cacao varieties that are resistant to pests and diseases are available for farmers, farming practices, phyto-sanitation and use of chemicals are some of the strategies adopted, however, with limited success. In collaboration with the National Research Institutes, an Industry-USDA-ARS sponsored global initiative on IPM has been initiated to address some of the problems in Latin America, Africa and Asia. The use of environmentally safe control agents, biological control and the rational use of insecticides and fungicides are the basis of this new joint program. Progress has been made in developing networks in countries affected by an insect, cocoa pod borer and the three fungal diseases, witches’ broom, frosty pod rot and black pod rot. Both basic and applied research, setting-up field trials and training both scientific personnel and farmers in farmer field schools are in progress. An overview of the current status will be presented.

Rice is the most important food crop in the developing countries. Crop losses due to insects, diseases, physiological and environmental factors can mount to tens of billions of dollars each year. Rice blast caused by Magnaporthe grisea and sheath rot caused by Rhizoctonia oryzae are the two most important pathogens in rice with rice blast accounting for 10-15% annual loss in the absence of chemical solutions. Current efforts to confer disease resistance in rice through transgenic expression of antimicrobial genes from different sources have met with various successes. In this work, unlike previous attempts where known insect antimicrobial peptides were used, a novel approach is taking place. We have used this novel approach to identify antimicrobial peptides with in vitro inhibitory activity against either M. grisea or R. oryzae. Fourth instar Manduca sexta were challenged with these pathogens to induce an immune response. Immune peptides showing antifungal activity against the pathogens were purified and their corresponding genes cloned. Several novel peptides have been generated by this approach. The gene, Mag-1 and Rhizoc-3 were transformed into rice biolistically and the To and T1 generations of the plants were generated and screened for enhanced disease resistance.

A severe Phytophthora disease wiped out entire crops at some nurseries and in landscapes of Virginia in 2003 and 2004. The disease started at the stem base and quickly collapsed the plant. It may also spread upwards along the stem. Infected roots were discolored. Diseased plants may defoliate. A total of 98 petunias representing eight cultivars were assessed in this study. Seedlings were purchased biweekly beginning in the first week of April and potted immediately with pine bark medium in new 2-liter containers at a local nursery. Potted plants were grown on the farm for 3 to 5 weeks then transported to the Virginia Tech’s Agricultural Research and Extension Center in Virginia Beach on May 3 and 24, and June 10 and 15, 2004, respectively. All plants were irrigated daily with well water and screened twice a week for disease symptoms. Typical disease symptoms were first observed in 4 plants on June 15. Isolation was performed from roots, bases and stems of diseased plants, resulting in 25 isolates of Phytophthora. All isolates had arachnoid mycelium and produced papillate sporangia. They also produced the single-strand conformation polymorphism pattern typical of Phytophthora nicotianae. The disease spread rapidly and caused 75% plant mortality by July 19. All cultivars appeared to be susceptible to this disease.

Propamocarb is a commonly used chemical for oomycete disease management. Resistance to this compound has been reported for species of Pythium but not Phytophthora. The objective of this study was to assess propamocarb sensitivity of Phytophthora nicotianae isolates from nursery crops and irrigation water. A total of 73 isolates including 2 recovered before propamocarb was marketed for fungicidal use was tested. Initial screening was conducted using clarified V8 juice agar amended with propamocarb at 1.8 or 10 mg ml(^–1). Approximately 44% of isolates were highly resistant (growing well in the presence of 10 mg ml(^–1)) and 33% were intermediately resistant. The effective concentration (EC(50)) was determined for 3 selected isolates from each group using the same medium amended at 0, 1, 10, 25, 50 or 100 mg ml(^–1). The average EC(50) values were 4.4 and 27.9 mg ml(^–1) for sensitive and resistant isolates, respectively. These isolates were further tested with geranium plants for sensitivity to propamocarb at 2.35 µl ml(^–1) (equivalent to the label-recommended rate 3 fl. oz./10 gal). Four to six days after inoculation, all isolates exhibited similarly high aggressiveness on water-treated seedlings, while resistant isolates consistently colonized propamocarb-treated seedlings. These results are consistent with those of in vitro assays.

Target spot is a damaging foliar disease of tobacco caused by Thanatephorus cucumeris that has become increasingly common in Virginia. Although periodic research has been conducted to identify effective control methods for this disease, neither resistant cultivars nor effective fungicides are available. Actigard significantly reduced target spot in trials conducted in 1995, 2001 and 2004, but disease pressure in 1995 and 2001 was relatively light. In 2004, application of Actigard, three rates of Quadris, or Rovral reduced target spot significantly compared to average losses of 23% in untreated control plots. Three applications of Actigard or Rovral reduced percent leaf area damaged on lower leaves from 23% to 2.1% and 6.8%, respectively. Applications of 0.5L/ha, 0.7L/ha, or 0.9L/ha Quadris reduced disease damage to 3.2%, 0.9% or 0.4%, respectively. Although differences in target spot severity were visually apparent and statistically significant, trends in overall yields among 2004 treatments did not mirror those in disease severity. This experiment will be repeated in 2005 to verify treatment effects on disease severity and to further examine the influence of target spot on flue-cured tobacco yield and quality.

The full-length beta-tubulin gene (tubA) coding sequence was PCR-amplified and sequenced from two economically important cereal Phaeosphaeria pathogens, P. nodorum (PN) and P. avenaria. A tubA gene of size 1,556 bp was identified in wheat- and barley-biotype PN, P. a. f. sp. avenaria, homothallic P. a. f. sp. triticea (Pat1) and one P. a. f. sp. t. isolate (Pat3), S-81-W10. Polymorphisms of the tubA gene were detected in two heterothallic P. a. f. sp. t. isolates (Pat2) from foxtail barley (Hordeum jubatum L.), the Phaeosphaeria spp. from dallis grass (Paspalum dilatatum) and two Polish PN isolates from rye. The size difference was due to the variation of intron length. All Phaeosphaeria isolates have identical 1,344 bp exons that can be translated into an identical 447 amino acid tubulin peptide (TUB), with the exception of the two heterothallic Pat2 isolates from foxtail barley. Six amino acid differences were evident in the TUB of these Pat2 isolates.

Automated Capillary Electrophoresis (ACE) can be used to characterize DNA fragments. This separation technique supports high throughput analysis for molecular procedures, to determine identity and relationships based on DNA differences. We have used Simple Sequence Repeat (SSR) markers in Theobroma cacao to identify germplasm in tropical collections, to demonstrate the limited genetic diversity of some collections, and to examine origins of accessions. SSR analysis is a straightforward procedure, and results are easily scored with ACE. However, generating markers by Amplified Fragment Length Polymorphism (AFLP) and analysis of results by ACE, are significantly more involved. Using AFLP, which requires no prior sequence information, we have successfully differentiated species, subspecies and populations of Erythroxylum coca. AFLP of fungal plant pathogens has demonstrated vegetative compatibility groups in Sclerotinia homoeocarpa and revealed population structure in Ophiosphaerella agrostis and in the Stemphylium species group. Issues in interpretation of AFLP analysis will be discussed.

Lilac has been reported as a host of the “sudden oak death” pathogen, Phytophthora ramorum, in England. It is a very important plant to the nursery industry in the U.S., so its susceptibility to ramorum blight is of interest. Seven accessions of lilacs (Syringa × prestoniae ‘Alexander’s Pink’, S. oblata ‘Betsy Ross’, S. × tribrida ‘Lark Song’, S. × laciniata, S. meyeri ‘Palibin’, S. pubescens ‘Miss Kim’ and S. vulgaris) were inoculated with a sporangial suspension (approx. 4000 sporangia/mL) and incubated in a dew chamber (20°C) for 4-5 days. Plants were rated for defoliation and disease (the number of infected leaves and percent leaf area affected). ‘Alexander’s pink’ was not very susceptible (only 0.5% infected leaves), and ‘Miss Kim’ was moderately susceptible, while all other lilacs tested developed large black lesions on 20-40% of leaves and suffered some defoliation.

Pear is an important deciduous fruit with high commercial value. However, pear production is often hampered by bacterial, fungal, and viral diseases. As part of our program aimed at improving resistance of pear crops to diseases via genetic engineering, we studied the influence of medium composition and growth regulators on adventitious shoot formation from in vitro propagated leaf explants of two popular commercial pear cultivars, ‘Onward’ and ‘Old Home’. Among three basal media (NN69, WPM, and C) tested, NN69 appeared to be the most suitable for promoting shoot regeneration. The cytokinin thidiazuron was much more effective than 6-benzylaminopurine in promoting shoot regeneration in both cultivars. The effect of two auxins, indole-3-butyric acid and alpha-naphthalene acetic acid, was genotype-dependent. Under optimal conditions, the regeneration frequency reached 90% and 100% for ‘Onward’ and ‘Old Home’, respectively. A comparative structural analysis revealed that shoot regeneration from ‘Onward’ leaf explants essentially occurred via direct organogenesis and that shoot regeneration from ‘Old Home’ leaf explants followed an indirect organogenesis pathway. The optimal regeneration conditions established in this study were successfully applied to Agrobacterium-mediated genetic transformation of the two pear cultivars.

Potato tubers with symptoms of soft rot were collected in nine different regions of Russia in 2004. More than 200 strains of Erwinia were isolated from over 200 samples. Based on their phenotypic properties, the strains were identified as either E. carotovora subsp. carotovora (Ecc) or E. carotovora subsp. atroseptica (Eca). In pathogenicity tests using potato seedlings, the Eca and Ecc strains caused large black linear and small, localized stem lesions, respectively. To determine their genetic diversity, RFLP analysis of the rec A gene (Waleron et al., 2002. Microbiology 148:583-595) was conducted for 32 and 20 typical Eca and Ecc strains, respectively. Results of the analysis were not able to differentiate between strains of Eca and Ecc. However, three groups containing both Eca and Ecc strains were evident. Group A produced a single 730 bp band and contained 15 Eca strains; group B produced an additional band over 1000 bp and contained 17 Eca and 16 Ecc strains; group C had no product and contained four Ecc strains. These results show that the Russian population of Ecc and Eca in potato contains considerable genetic diversity.

Symptoms of general stunting and yellowing of leaves were observed in diseased cultivated strawberry (Fragaria × ananassa Duch.) in the Anyksciai region of Lithuania. Analysis of 16S ribosomal (r) DNA amplified in the polymerase chain reaction (PCR) indicated that the symptoms were associated with infection by a phytoplasma, designated strawberry yellows (StrawY) phytoplasma. Phylogenetic analysis of 16S rDNA sequences revealed that StrawY phytoplasma, ‘Candidatus (Ca.) Phytoplasma (P.) australiense’, ‘Ca. P. solani’, ‘Ca. P. asteris’, and ‘Ca. P. japonicum’ shared a common ancestor but are distinct from each other. Although the StrawY phytoplasma appeared most closely related to ‘Ca. P. japonicum’, the results from rDNA analysis, in addition to clear differences in natural hosts and geographical distribution, indicated that StrawY phytoplasma is a novel phytoplasma species.

Cyclic hydroxamic acids are secondary metabolites found in the Poaceae and are implicated in the allelopathy of rye (Secale cereale), but the toxicity of these compounds against plant-parasitic nematodes is unknown. DIBOA (2,4-dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one) and DIMBOA (2,4-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one) and their degradation products BOA (benzoxazolin-2(3H)-one) and MBOA (6-methoxy-benzoxazolin-2(3H)-one) were screened against Meloidogyne incognita second-stage juveniles (J2) and eggs. Eggs were less sensitive to the hydroxamic acids than J2. DIBOA was the most toxic acid tested. It caused a 50% reduction in egg hatch with a lethal concentration (LC(50)) of 74 µg/ml. The LC(50) was 21 µg/ml for J2. When M. incognita J2 were exposed to DIBOA for 48 h and the compound was removed and replaced with water, the LC(50) increased to 40.7 µg/ml. For DIMBOA the LC(50) remained the same after rinse, 46.1 and 43.1 µg/ml before and after rinse, respectively. MBOA was not toxic to M. incognita eggs, but was toxic to M. incognita J2 (LC(50)s of 44 and 20 µg/ml before and after water rinse). BOA was the least toxic hydroxamic acid tested, and did not reduce M. incognita egg hatch after one week of exposure.

Hairy vetch green manure is a promising alternative management approach for Fusarium wilt of watermelon, but is insufficient when triploid watermelon is grown in severely infested soils. A field experiment in a split-split-plot design was conducted over two years to evaluate efficacy of hairy vetch green manure alone and in combination with a moderately wilt-resistant triploid cultivar for wilt suppression compared with preplant soil fumigants. An incorporated hairy vetch winter cover crop was as effective as the resistant cultivar in reducing wilt incidence and increasing fruit yield. The additive effect that was observed when both treatments were combined was greater than that obtained with the fumigants methyl bromide or metam sodium. There was a trend toward increased soil populations of Fusarium oxysporum following green manure. Stem colonization by the pathogen was lowest in the resistant cultivar grown in manured plots. The combined use of hairy vetch green manure and a resistant cultivar can enhance suppression of Fusarium wilt in triploid watermelon.